So far, generation of broadly neutralizing antibody responses against diverse primary HIV isolates has been an elusive target. Therefore, the aim of this project is to design and evaluate novel Env immunogens for their ability to induce broadly neutralizing antibody responses against diverse primary isolates. We previously reported that an Env immunogen (o-gp140SF162DV2) containing a partial deletion in the second variable loop (V2) derived from SF162 (R5 tropic), when used in a DMA prime-protein boost regimen in rhesus macaques, induced neutralizing antibodies against some heterologous subtype B primary isolates as well as protection to the vaccinated animals upon challenge with pathogenic SHIVSF162P4. We plan to continue working on trimeric Env and use four complementary approaches to further enhance the exposure of conserved neutralizing epitopes to improve the efficacy of Env immunogens in inducing broadly neutralizing antibodies and protection in challenge model. In the first, we propose to increase the exposure of conserved neutralizing epitopes involved in receptor and co-receptor binding regions of the Env by introducing deletions in V-regions and "bridging-sheet" following rational and random approaches. In the second, we seek to expose neutralizing epitopes by introducing site specific or global deglycosylation either alone or in combination with b-sheet and/or V- loops. In the third, we seek to expose these epitopes by the use of Env complexed to rationally designed CD4 mimics or other scaffolds. Lastly, we propose to express Env trimer in non-glycosylated form to enhance the exposure of critical neutralizing epitopes that are shielded by extensive glycosylation. We will use subtype B antigens as a model for evaluating the efficacy of various approaches mentioned above. The best approach will be used for optimizing Env antigens from other subtypes e.g. A and C. We will screen candidate immunogens for expression, CD4-binding, and binding to a panel of monoclonal antibodies of known specificities. Once a candidate has met pre-defined criteria, it then will be advanced to immunogenicity studies in rabbits. Neutralizing antibody and cellular responses induced by the lead candidate will be optimized using adjuvants, formulations, and vaccine regimens. The best Env immunogen will be advanced to vaccine/challenge studies in primates. These studies should yield important information for the design of next generation HIV vaccines for future clinical evaluations.